WO2007032482A1 - Polymorphisme genetique utilise pour prevoir une reponse a un agent antidepresseur - Google Patents

Polymorphisme genetique utilise pour prevoir une reponse a un agent antidepresseur Download PDF

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WO2007032482A1
WO2007032482A1 PCT/JP2006/318386 JP2006318386W WO2007032482A1 WO 2007032482 A1 WO2007032482 A1 WO 2007032482A1 JP 2006318386 W JP2006318386 W JP 2006318386W WO 2007032482 A1 WO2007032482 A1 WO 2007032482A1
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gene
polymorphism
antidepressant
reactivity
subject
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PCT/JP2006/318386
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English (en)
Japanese (ja)
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Junichi Azuma
Tsuyoshi Fukuda
Megumi Yamashita
Yuka Hosoi
Masaki Kato
Masataka Wakeno
Yoshiteru Takekita
Gaku Okugawa
Toshihiko Kinoshita
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The New Industry Research Organization
Osaka University
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Priority to JP2007535557A priority Critical patent/JP5002746B2/ja
Publication of WO2007032482A1 publication Critical patent/WO2007032482A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a method for predicting reactivity to an antidepressant based on a genetic polymorphism of a subject.
  • the present invention provides a technique useful for testing and diagnosis in pharmacotherapy for the purpose of improving depression.
  • depression is a familiar disease, as “depression is a cold of the heart”.
  • the major biological hypotheses for the development of depression include monoamine (serotonin, noradrenaline) nervous system dysfunction, hypothalamic-pituitary-adrenal (HPA) dysfunction (stress adaptive dysfunction), ecological rhythm abnormality, Although neuroplasticity abnormalities have been envisaged, they have not yet been confirmed. Since the prevalence of depression relatives is higher than in general subjects, there is no doubt that some genetic predisposition exists in the pathogenesis. Depression can be regarded as some kind of stress vulnerability, and its foundation includes monoamine nervous system, HPA system and neuroplasticity abnormalities, which are excessively affected by stress and the stress adaptation mechanism in the brain has collapsed. It is assumed that the disease will occur.
  • SSRI is used as a first-line drug for treating depression, and it is known that there are great individual differences in the clinical effect.
  • SSRI is an abbreviation for Selective Serotonin Reuptake InWbitors, a new generation of a group of drugs that selectively inhibit serotonin reuptake via serotonin transporters at nerve terminals. It is a name that refers to a depressant.
  • SNRI can be cited as a new generation of antidepressants.
  • SNRI is an abbreviation for serotonin & noradrenaline reuptake inhibitors (Selective) Serotonin & Noradorenaline Reuptake InWbitors, and is a name that refers to a group of substances that selectively inhibit serotonin and noradrenaline reuptake.
  • Non-Patent Document 2 was published by the present inventors, and the clinical effects and genetic polymorphisms of patients with two typical antidepressant drugs paroxetine (paroxetine) and fluvoxamine (fluvoxamine) belonging to SSRI. We reported that the clinical effect of paroxetine was higher than that of fluvoxamine when the genotype of the LPR polymorphism was s / s.
  • TPH2 Noriant tributofan hydroxylase-2
  • TPH3 tributophane hydroxylase
  • Non-patent Document 4 A polymorphism that is present in an exon of the TPH2 gene and that reduces serototone production by 80% in vitro has been reported (Non-patent Document 4 below). Many mutations were detected in patients with depression, suggesting that this mutation is an important region for TPH2 enzyme activity and may be involved in the development of depression.
  • Non-Patent Document 8 ⁇ 9 below There has been a report that there is a correlation between polymorphisms related to the TPH2 gene and attention deficit hyperactivity disorder (ADHD)! / Talk.
  • Non-patent document 1 Prog Neuropsychopharmacol Biol Psychiatry. 2005,29: 1062-1073
  • Non-patent document 2 Int Clin Psychopharmacol. 2005,20: 151-156
  • Non-Patent Document 3 Science. 2003,299: 76
  • Non-Patent Document 4 Neuron. 2005, 45: 11-16
  • Non-Patent Document 5 Mol Psychiatry. 2004,9: 980-983
  • Non-Patent Document 6 Mol Psychiatry. 2004,9: 1030-1036
  • Non-Patent Document 7 Psychiatry Res. 2005,134: 195-198
  • Non-Patent Document 8 Mol Psychiatry. 2005 Oct; 10 (10): 944-9.
  • Non-Patent Document 9 Mol Psychiatry. 2005 Dec; 10 (12): 1126- 32.
  • the present invention has been made in view of the above-mentioned problems, that is, the importance of predicting antidepressant drug reactivity before administration, and the object thereof is the polymorphism of a subject. It is to provide a method for predicting reactivity to antidepressants based on the above.
  • the present inventors have conducted intensive research assuming the involvement of genetic factors in the cause of large individual differences in the clinical effects of antidepressants, and so far, serotonin, which is the site of action of the antidepressant SSRI.
  • serotonin which is the site of action of the antidepressant SSRI.
  • the LPR polymorphism of the transporter is one of the causes.
  • TPH2 tributophan hydroxylase-2
  • gene polymorphisms present in the promoter region of the TPH2 gene are useful for predicting individual differences in the clinical effects of antidepressants, and the gene polymorphisms are used to accurately predict responsiveness to antidepressants. It was also found that, by combining with other genetic polymorphism test results such as the above-mentioned LPR polymorphism, it becomes possible to predict the response more finely and contribute to the formulation of treatment plans suitable for individual patients.
  • D2 receptor D2 receptor
  • the inventors have found that the TaqlA polymorphism of the gene and the TCAT recombinant polymorphism of the tyrosine hydroxylase (TH) gene are useful for predicting the reactivity to antidepressants, and have completed the present invention. .
  • the present invention includes the following inventions A) to A) as industrially useful inventions.
  • TPH2 tryptophan hydroxylase-2
  • D2 dominine D2 receptor
  • TH tyrosine hydroxylase
  • tryptophan hydroxylase-2 (tryptophan hydroxylase-2) is also called tryptophan hydroxyloxygen type 2 and is considered to be an enzyme responsible for the rate-limiting step of serotonin biosynthesis in the brain.
  • this enzyme is abbreviated as “TPH2” and its gene is referred to as “TPH2 gene”.
  • Dopamine D2 receptor is one of the subtypes of dopamine receptor.
  • this receptor is abbreviated as “DRD2”, and the gene is referred to as “DR D2 gene”.
  • DR D2 the gene is referred to as “DR D2 gene”.
  • TH the gene is referred to as “TH”. It is called “gene”.
  • the present invention examines TPH2 gene, DRD2 gene, TH gene, or polymorphisms in the vicinity of them in the genome. In other words, this is at least one polymorphism associated with these genes. Means to inspect. It is not limited to the examination of polymorphisms present in the exon region or intron region of these genomic genes, but is intended to examine polymorphisms present in transcription regulatory regions such as promoter regions, control regions or untranslated regions. May be.
  • “gene polymorphism” includes a polymorphism existing in such a promoter region and the like, and has a broad meaning of a polymorphism related to the gene.
  • the present invention it is preferable to use a method in which a plurality of polymorphisms related to any of the above three genes are examined and the reactivity of a subject to an antidepressant is predicted based on the results.
  • paroxetine praroxetine
  • fluvoxamine fluvoxamine
  • fluoxetine fluoxetine
  • sertraline sertraline
  • dexfenfluramine dexfenfluramine
  • examples of the above-mentioned antidepressant SNRI include milnacipran and the like, in addition to venlafaxine and duloxetine.
  • the present invention is suitable for predicting the reactivity against the above-mentioned antidepressant drug S SRI or SNRI, but other antidepressants having a serotonin reuptake inhibitory effect (tricyclic antidepressants, tetracyclic antidepressants). It can also be used for predicting the reactivity to depression and the like.
  • C) By detecting at least one of the polymorphism present in the promoter region of the TPH2 gene, the TaqlA polymorphism of the DRD2 gene, and the TCAT repeat polymorphism of the TH gene, The method according to A) or B) above, wherein the reactivity to an antidepressant is predicted.
  • the present inventors have made a polymorphism present in the promoter region of the TPH2 gene (r si 1178997 gene polymorphism), a TaqlA polymorphism in the DRD2 gene (rsl800497 gene polymorphism), or a TCAT repeat polymorphism in the TH gene.
  • r si 1178997 gene polymorphism a polymorphism present in the promoter region of the TPH2 gene
  • rsl800497 gene polymorphism a TaqlA polymorphism in the DRD2 gene
  • TCAT repeat polymorphism a polymorphism present in the promoter region of the TPH2 gene
  • TCAT tetranucleotide
  • a serotonin transporter gene, a serotonin receptor gene, or a polymorphism existing in the vicinity thereof in the genome is examined, and the results are taken into consideration to determine whether the subject has an anti-depressant drug.
  • the above-mentioned “polymorphism existing in the vicinity thereof” means, in other words, a polymorphism related to the serotonin transporter gene (or serotonin receptor gene), that is, A polymorphism present in a transcriptional regulatory region such as a promoter region, a regulatory region, or a non-translated region may be examined.
  • the genetic polymorphism test comprises the steps of amplifying the gene region sandwiching the polymorphic site by converting the genomic DNA prepared from the subject into a saddle type. And genotyping based on the amplified fragment.
  • TPH2 tryptophan hydroxylase-2
  • D2 dopamine D2 receptor
  • TH tyrosine hydroxylase
  • reactivity to an antidepressant can be easily and quickly predicted objectively based on TPH2, DRD2, and TH gene polymorphisms.
  • disease treatment it can be suitably used for predicting patient responsiveness to antidepressants.
  • antidepressants especially SSRI
  • the clinical effects of antidepressants, especially SSRI vary greatly from person to person, and the severity of depression is likely to lead to suicide attempts. It was strongly desired to predict the drug objectively at the early stage of treatment and to carry out appropriate drug treatment suitable for individual patients.
  • the present invention makes it possible to predict the antidepressant drug response of a patient with high accuracy before administration, realize effective and safe depression treatment, and contribute to improvement of medical quality and improvement of treatment efficiency. Is.
  • FIG. 1 is a graph showing the results of examining the correlation between the TPH2 gene polymorphism (rslll78997 polymorphism) and the reactivity to the antidepressant SSRI.
  • Fig. 2 shows the results of investigating the correlation between the TPH2 gene polymorphism (rslll78997 polymorphism) and the antidepressant SSRI reactivity in the sZs group with the LPR polymorphism of the serotone transporter gene It is a graph.
  • FIG. 3 is a graph showing the results of examining the correlation between the above TPH2 gene polymorphism (rsl 1178997 polymorphism) and the above LPR polymorphism and the reactivity to the antidepressant SSRI.
  • FIG. 4 is a diagram illustrating polymorphisms on the DRD2 gene.
  • FIG. 5 is a graph showing the results of examining the correlation between TaqlA polymorphism (rsl800497 polymorphism) of the DRD2 gene and reactivity to the antidepressant drug SSRI.
  • FIG. 6 is a diagram illustrating polymorphisms on the TH gene.
  • FIG. 7 is a graph showing the results of examining the correlation between the TCAT repeat polymorphism of the TH gene and the reactivity to the antidepressant SSRI (paroxetine).
  • FIG. 8 is a graph showing the results of examining the correlation between the TCAT repeat polymorphism of the TH gene and the reactivity to the antidepressant SSRI (fulvoxamine).
  • FIG. 1 is a graph showing that a significant correlation was observed between this rsl 1178997 polymorphism and reactivity to the antidepressant SSRI.
  • the analysis method is roughly as follows. In other words, 100 patients (mean age 43.7 ⁇ 14.9) who were diagnosed as major depression or dysthymic disorder and had written consent were used, and no SSRI paroxetine or fluvoxamine was administered by envelope method. Assigned to work. The drug dose started at 20 mg and 50 mg, respectively, and could be increased to 40 mg and 150 mg, respectively, depending on the degree of symptom improvement. The clinical effect was evaluated over time by the Hamilton Depression Rating Scale (HAM-D) score, which is a scale of depression. The graph in Figure 1 shows fluvoxamine. This is the result of 41 patients taking depression. The lower the “HAMD change rate (%)” on the vertical axis of the graph, the higher the improvement rate by the drug, that is, the higher the clinical effect.
  • HAM-D Hamilton Depression Rating Scale
  • the ⁇ / ⁇ possession group that has the homology of the rsl 1178997 polymorphic allele in the rsl 1178997 polymorphism is heterozygous for the ⁇ and ⁇ alleles.
  • the clinical effect of fluvoxamine was higher.
  • the early stage force of the second week of drug administration was significantly better.
  • no “ ⁇ / ⁇ ” holders with the “ ⁇ ” allele were detected.
  • “1/2”, “3/4”, and “5/6” indicate 1-2 weeks, 3-4 weeks, and 5-6 weeks, respectively, and “ ⁇ ” indicates the number of patients in each group. *, “#” And “+” indicate the ⁇ value of t test (the same applies to other graphs).
  • the rsl 1178997 polymorphism was shown to be significantly correlated with the reactivity to the SSRI drug fluvoxamine. Based on these results, the rsl 1178997 polymorphism of the depression patient was examined, and if the base of the polymorphic site is a genotype ⁇ / ⁇ that has a homology with the allele of ⁇ T '', SSRI Reactivity can be predicted to be an effective responder. On the other hand, in the case of genotype “T / A” or “A / A” where the base of the polymorphic site has an allele of “A”, a non-responsor (non-responsor) having no effect on SSRI.
  • the der can be predicted to be a force responder or a poor responder.
  • the patient's responsiveness to the antidepressant ie, whether the patient is a responder (or poor responder) is predicted before administration. This can contribute to efficient and appropriate treatment of depression.
  • the present invention is a method for predicting a subject's antidepressant responsiveness based on the test result of the above-mentioned TPH2 gene polymorphism (rsl 1178997 polymorphism). It is not limited to direct inspection methods. For example, by testing for other polymorphisms on the TPH2 gene that are linked to the rsl 1178997 polymorphism and form a haplotype, the rs 11178997 polymorphism can be indirectly tested to determine the anti-depressant drug reactivity. Can be predicted. In addition, since this analysis revealed a correlation between TPH2 gene polymorphism and antidepressant responsiveness, it was based on other gene polymorphisms present on the TPH2 gene! It is also possible to predict responsiveness.
  • the rsl 1178997 polymorphism is completely linked to the 11178998 polymorphism (-52 / on the TPH2 gene, and when the rsl 1178998 polymorphism is the “A” allele, the rsl 1178997 polymorphism is the “T” allele.
  • the rsl 1178997 polymorphism test may be tested with other linked mutations.
  • the gene polymorphism is not limited to a single nucleotide polymorphism, for example, a polymorphism due to the presence or absence of a defective portion, that is, consisting of two or more bases Difference between insertion type with specific base sequence and deletion type lacking this base sequence It may be a genotype having The present invention is not limited to single nucleotide polymorphisms, and may predict antidepressant drug reactivity based on gene polymorphisms based on the presence or absence of such a defective portion, or may be antitumor based on other gene polymorphisms such as rearrangement. Depressant drug reactivity may be predicted.
  • the TPH2 cDNA sequence is Since it is registered and published under the session number “NM_173353” etc., it may be designed with reference to the nucleotide sequences disclosed in these databases (especially the gene polymorphism of the exon region or intron region of the TPH2 gene). If you want to inspect).
  • PCR-SSCP single strand conformation polymorphism
  • the PCR reaction solution (201) was prepared by adding 60 ng of genomic DNA extracted from the patient's peripheral blood force to the optimal buffer, enzyme, dNTP (0.2 mM each), forward primer (0.5 ⁇ ) and reverse primer ( The composition contained 0.5 ⁇ ).
  • the sequences of the designed forward primer ( ⁇ 2-473F) and reverse primer ( ⁇ 2 -473R) are as shown in (a) and (b) below.
  • As PCR reaction conditions 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 30 seconds were used as one cycle, and DNA was amplified by performing this step for 35 cycles.
  • the obtained PCR products were separated by a method called SSCP (single strand conformation polymorphism), and the gene difference was determined by the electrophoresis pattern.
  • the PCR product was first diluted 4-fold with Tris buffer, and 3 U of this sample was added to the denaturing solution 6 / zL.
  • the denaturing solution is 94% formamide, 0.05% xylene cyanol blue.
  • Solution a solution of 0.04% bromophenol blue.
  • the PCR product was heat denatured by treatment at 95 ° C for 5 minutes. After electrophoresis using GeneGel Excel 12.5 / 24 kit (Amersham), silver staining was performed.
  • FIG. 4 shows the power of representative polymorphisms of DRD2.
  • -141C Ins / Del polymorphism in the promoter region, 3, TaqlA polymorphism in the UTR region (rsl800497 polymorphism), and exon 7 Ser311Cys polymorphism has been studied in many diseases.
  • FIG. 5 is a graph showing that as a result of the analysis by the present inventors, a significant correlation was observed between the Taq 1 A polymorphism and the reactivity to the antidepressant drug S SRI. .
  • the analysis method is the same as in the case of TPH2, and the graph in FIG. 5 shows the results of 41 patients with depression who took fluvoxamine. The lower the “: HAMD change rate (%)” on the vertical axis of the graph, the lower the improvement rate by the drug, that is, the clinical effect Is rated high.
  • the A2 allele possession group that is, the “A1 / A2” and “A2” alleles having the “A1” and “A2” alleles in heterogeneity are shown.
  • “A2 / A 2” with homo is more effective in the clinical phase of fluvoxamine than the “A1 / A1” group with homo A1 allele. Obtained.
  • “ ⁇ ” indicates that there was a significant difference between the A2 allele possession group and the A1 allele homology group in repeated measure ANOVA (the same applies to FIGS. 7 and 8). It shows that there was a significant difference in the analysis).
  • TaqlA polymorphism was shown to significantly correlate with the responsiveness to the SSRI drug fluvoxamine. Based on these results, TaqlA polymorphisms in depressed patients are examined, and if the polymorphism is in the A2 allele possession group (“A1 / A2” or “A2 / A2”), it shows an effect on SSRI Reactivity can be predicted to be a responder. On the other hand, if the polymorphism is the A1 allele homozygote group “A1 / A1”, it is predicted to be a non-responder that has no effect on SSRI or a poor responder (poor responder). be able to.
  • testing TaqlA polymorphism alone also shows the patient's responsiveness to antidepressants (therapeutic responsiveness), ie whether the patient is a responder (or poor responder). Predicting before administration can contribute to efficient and appropriate treatment of depression.
  • PCR-RFLP Restriction Fragment Length Polymorphism
  • the PCR reaction solution (201) was prepared by adding 30 ng of genomic DNA extracted from the patient's peripheral blood to the optimal buffer, enzyme, dNTP (each 0.2 mM), forward primer (0.5 ⁇ 0.5) and reverse primer ( The yarn was 0.5 ⁇ ⁇ ).
  • the sequences of the designed forward primer (D2-TaqlA F) and reverse primer (D2-TaqlA R) are as shown in (a) and (b) below.
  • PCR reaction conditions were 94 ° C for 30 seconds, 58 ° C for 30 seconds, and 72 ° C for 30 seconds, and DNA was amplified in steps of 35 cycles.
  • the Taql A polymorphism inspection method is not limited to the above method.
  • TaqlA polymorphisms are indirectly tested by examining other gene polymorphisms on the DRD2 gene that are linked to TaqlA polymorphisms to form haplotypes. The reactivity of an antidepressant may be predicted.
  • this analysis found a correlation between D RD2 gene polymorphism and antidepressant drug reactivity, it was determined that there was an antidepressant responsiveness based on other gene polymorphisms present on the DRD2 gene. Can also be predicted.
  • the base sequence of 201 nucleotides in length including the TaqlA polymorphic site is shown below.
  • the TaqlA polymorphism site is located at the 101st position, the A1 allele is “TJ (thymine)”, and the A2 allele is “C” (cytosine).
  • probes and primers can be designed based on this sequence information, and the polymorphism can be examined.
  • TCAT TH tetranucleotide repeat polymorphism
  • TH tyrosine hydroxylase
  • FIG. 6 shows a representative gene polymorphism of TH.
  • the above TCAT repeat polymorphism in intron 1 is most frequently studied in relation to various diseases.
  • the other is the Val81Met polymorphism of amino acid substitution, which has been correlated with schizophrenia (Neuropsychiatric Genetics. 2003, 116B: 23-26, Molecular Psychiatry. 2001, 6:31 5-319).
  • TCAT repeat polymorphism activation of the noradrenergic nervous system (Hypertension. 1999, 32: 676-682, Life Science. 1997, 61: 1341-1347) and reduced extroversion in healthy individuals (Neuroscience Research. 2006, 54 : 180-185).
  • TCA T repeat polymorphism has not been reported to be associated with antidepressant responsiveness.
  • the TCAT repeat polymorphism has 6 to 10 repeats (T6 to T10) alleles, 9 repeats for Japanese and 10 repeats for Caucasian.
  • FIG. 7 and FIG. 8 are graphs showing that a significant correlation was observed between the TCAT repeat polymorphism and reactivity to the antidepressant drug SSRI.
  • the analysis method is the same as for TPH2 and DRD 2, but the graph in Fig. 7 shows the results for 39 patients with depression who took paroxetine, while the graph in Fig. 8 shows the patients with depression who took fluvoxamine. The result of the name.
  • the TCAT repeat polymorphism is significantly correlated with reactivity to the antidepressant SSRI. Based on these results, TCAT repeat polymorphisms in depressed patients were examined, and if the polymorphism was in the ⁇ 9 + / 9 + '' group with 9 repeat alleles homozygous, it was more fully than paroxetine. It can be predicted that taking voxamine (or antidepressants with similar trends) will be more responsive to the patient. On the other hand, when the polymorphism has an allele other than 9 repeat alleles (“9 + / 9-” or “9- / 9-”), paroxetine (or a similar trend is shown) rather than fluvoxamine.
  • the TCAT repeat polymorphism was examined and determined using PCR as follows. First, for the PCR method, the PCR reaction solution (20 ⁇ 1) was prepared by adding 60 ng of genomic DNA extracted from the patient's peripheral blood, optimal buffer, enzyme, dNTP (each 0.2 mM), forward primer ( ⁇ ⁇ ), and reverse. A yarn containing a primer (1 mm) was formed. The sequences of the designed forward primer (TH 1F) and reverse primer (TH 1R) are as shown in (a) and (b) below. As PCR reaction conditions, 94 ° C for 30 seconds, 58 ° C for 30 seconds, and 72 ° C for 30 seconds were used as a cycle, and DNA was amplified by performing 40 cycles of this cycle.
  • the obtained PCR product was electrophoresed using a capillary and the difference of 4 bp was observed and judged.
  • TCAT repeat polymorphisms are indirectly detected by examining other gene polymorphisms on the TH gene that are linked to TCAT repeat polymorphisms to form haplotypes.
  • the mold may be examined to predict antidepressant responsiveness.
  • TH gene polymorphism and antidepressant responsiveness was found in this analysis, so antidepressant responsiveness was determined based on other gene polymorphisms present on the TH gene. It is also possible to predict this.
  • This sequence is a 9-repeat allele sequence, and the site in Katsuko corresponds to the polymorphic site.
  • a probe or primer can be designed based on this sequence information, and the polymorphism can be inspected.
  • the present invention examines at least one polymorphism related to the TPH2 gene, DRD2 gene or TH gene and predicts the reactivity to an antidepressant.
  • a plurality of gene polymorphisms related to these genes are used.
  • the method of predicting responsiveness and predicting reactivity to antidepressants based on the test results is a preferred method, and similarly, combining gene polymorphisms related to these genes with polymorphisms of other genes Thus, a method for predicting reactivity to an antidepressant is also preferred and a method.
  • Fig. 3 shows the result of examining the correlation between the TPH2 gene polymorphism (rslll78997 polymorphism) and the LPR polymorphism test results and the patient's response to the antidepressant fluvoxamine It is a graph.
  • wild and hetero indicate that the genotype of the rslll78997 polymorphism is “T / T” and “ ⁇ / ⁇ ”, respectively, and ⁇ / sj “1/1” and “s / s” are LPR. Indicates the polymorphic genotype.
  • the combination of “1/1” and “wild” has the highest therapeutic response, whereas the combination of “s” and “heter 0 ” has the lowest therapeutic response. I helped.
  • Each combination of “s / s” and “wild”, ⁇ / sj and “hetero”, “l / s” and “wild” shows intermediate therapeutic response between them, and has a certain effect on SSRI.
  • the reactivity can be predicted to be the same as the bonder shown. In this way, by combining the test results of a plurality of gene polymorphisms, it becomes possible to predict the reactivity more finely.
  • HTR serotonin receptor
  • the sequence described under each gene polymorphism of (1) to (6) above is a base sequence in the vicinity of the front and back including the polymorphic site, and the site in Katsuko corresponds to the polymorphic site. To do. These base sequences are also described in SEQ ID NOs: 11 to 16 in the sequence listing, respectively, but in the sequence listing, the base at the polymorphic site is represented by one letter by the universal code.
  • the gene polymorphism in (6) above is a polymorphism due to the presence or absence of a defective portion, and SEQ ID NO: 16 describes the nucleotide sequence of the insertion type (ins).
  • the gene polymorphism (HTR2A-1438A / G) in (3) above is a number when converted into a reverse-complemented strand, and thus is T / C in the above sequence (the same applies to SEQ ID NO: 13).
  • Serotonin receptor (HTR) gene polymorphisms (1) to (6) above and anti-depressant SSRI The specific correlation with the treatment responsiveness will be described as follows.
  • the polymorphism in (4) (HT R2A 102T / C: rs6313) was completely linked to the polymorphism in (3) above (linkage disequilibrium), so the C / C group tended to have a high improvement rate. Admitted.
  • the improvement rate tends to be high in the C / C group.
  • the polymorphism (HTR3B-100_-102AAG ins / del), the ins / A tendency toward a high improvement rate was observed in the ins group.
  • paroxetine clinical effects and two gene polymorphisms were analyzed by combining the two polymorphisms, and the odds ratio was 15.5. The prediction rate improved.
  • a method of predicting the reactivity to an antidepressant by combining a plurality of gene polymorphisms is a preferable method.
  • the predictive method based on discriminant analysis and the predictive method based on decision trees as disclosed in the specification of International Application No. PCTZJP2006Z312519 are preferable when predicting reactivity by combining test results of multiple gene polymorphisms. It is.
  • each prediction method described above is performed using a program.
  • the present invention includes such an antidepressant drug reactivity prediction support program. That is, the prediction support program of the present invention is (1) 1 or 2 related to any one of TPH2, DRD2 and TH.
  • the program of the present invention can be provided as a computer-readable recording medium on which the program is recorded.
  • Examples of such recording media include magnetic storage media such as flexible disks, hard disks, and magnetic tapes, and optical storage media such as CD-ROM, CD-R, CD-RW, DVD-ROM, DVD-RAM, and DVD-RW.
  • Powers that can exemplify electrical storage media such as RAM and ROM, and magnetic Z optical storage media such as MO are not limited to these.
  • methods for investigating TPH2, DRD2, and TH gene polymorphisms and other gene polymorphisms are not particularly limited, and can be used to directly or indirectly examine polymorphisms on genes.
  • Known methods can be applied, and methods developed in the future may be used.
  • the method used for this analysis to examine gene polymorphisms by PCR is a simple method with good accuracy, so this method is briefly described below.
  • the DNA sample to be used for the examination is any organ 'tissue' cell of the subject (including blood, cells in amniotic fluid, cells cultured from the collected tissue, etc.) 'Extract. As long as gene amplification by PCR is possible, the DNA purification and extraction step may be omitted or simplified.
  • PCR is performed using the genomic DNA in the DNA sample prepared by the above method as a saddle, and the polymorphic site is sandwiched. Amplify the gene region. Then, based on the obtained amplified fragment, the length of the amplified fragment can be examined by Primer extension method (primer extension method), PCR-RFLP method, or electrophoresis. Determine genotype by methods.
  • each condition and reagents used in the PCR method are not particularly limited.
  • genetic polymorphism testing may use methods other than PCR. If the polymorphism on the gene can be tested directly or indirectly, a method for testing the base in a single nucleotide polymorphism (SNP) (SNP typing), or a polymorphism based on the presence or absence of a defective part
  • SNP single nucleotide polymorphism
  • SNP typing single nucleotide polymorphism
  • Various conventionally known methods such as the method can be applied (for example, refer to the document “Post-Sequence Genomic Science (1) SNP gene polymorphism strategy” (Nakayama Shoten)).
  • a testing method using a genetic polymorphism testing instrument such as a DNA chip can be mentioned.
  • This method uses a DNA chip (or similar instrument) with a probe for testing TPH2, DRD2 and TH gene polymorphisms, or a probe for testing other gene polymorphisms on the substrate.
  • This is a method of performing gene typing using this DNA chip or the like based on the presence or absence of a hybridization signal between a gene sample from a subject and a probe.
  • an adjacent nucleotide sequence including the base of the polymorphic site for example, about 8 to 30 nucleotides in length
  • its complementary sequence is used.
  • DNA chip mainly refers to a synthetic DNA chip that uses synthesized oligonucleotides as probes. It also uses an affixed DNA microarray that uses cDNA such as PCR products as probes. Good.
  • a DNA chip containing such probes for TPH2, DRD2 and TH gene polymorphism assays can be used as a reactivity prediction chip for the antidepressant SSRI.
  • a point mutation detection method such as PCR-SSCP method or Allele specific PCR method may be used, or an amplification method other than PCR method (for example, RCA method). Etc.) may be used.
  • the base sequence of the amplified fragment can be directly determined using a nucleotide sequence determination device (Sequencer), and then the gene can be typed.
  • genetic polymorphism testing may be performed based on polymorphisms present in intron sequences, control sequences, etc., in addition to coding sequences encoding proteins.
  • the polymorphism (mutation) is a mutation on the coding sequence
  • the polymorphism (mutation) can be detected based on cDNA prepared from RNA or mRNA.
  • the polymorphism (mutation) may be detected from the amino acid sequence of the protein.
  • the gene polymorphism testing kit of the present invention is a primer designed based on these gene sequences or their neighboring sequences in order to directly or indirectly test TPH2, DRD2 and TH gene polymorphisms.
  • it contains probes, (1) Enzymes and reagents used for sample preparation, (2) Enzymes and reagents used for reverse transcription reaction, (3) Enzymes and reagents used for PCR method, ( 4) It may contain 1 or 2 or more of enzymes and reagents used to determine the base sequence! /.
  • TPH2, DRD2 and TH gene polymorphisms and antidepressant responsiveness shows the expression of TPH2, DR D2 and TH based on these gene polymorphisms. It can be said that individual differences in the amount (or activity, etc.) affect the antidepressant responsiveness. Therefore, by developing TPH2, DRD2 or TH as a target (drug discovery target) and searching for substances that affect the expression level, activity, binding to other substances, etc., development of efficient antidepressants, In particular, it can be expected to develop drugs that can be used in combination with antidepressants such as SSRI to improve their efficacy.
  • the present invention includes such an antidepressant screening method targeting TPH2, DRD 2 and TH.
  • screening method of the present invention various conventionally known methods for examining the expression level of gene 'protein, changes in protein activity, binding between molecules, etc. can be applied, and are not particularly limited. . Further, a screening method newly developed after the present invention may be used. Either in vitro or in vivo screening systems may be used, and screening may be performed with a cell-free system. In addition to the human-derived gene'protein used for screening, a mouse-derived or other animal-derived protein may be used. Of course, you may screen using the information regarding the three-dimensional structure of protein.
  • the present invention relates to a method for predicting the reactivity of a subject to an antidepressant based on genetic polymorphisms of TPH2, DRD2, and TH. It can be used for testing, diagnosis, etc. in the treatment of depression using drugs, especially SSRI.

Abstract

La présente invention concerne une étude effectuée afin de déterminer une corrélation entre des polymorphismes génétiques dans la tryptophane hydroxylase-2 (TPH2) et la réponse à un agent antidépresseur. Grâce à cette analyse, un polymorphisme génétique utile pour prévoir la réponse à un antidépresseur peut être trouvé dans une région de promoteur du gène TPH2, ce qui permet de prévoir la réponse à un antidépresseur de manière assez précise en utilisant le polymorphisme génétique. On a également découvert que les polymorphismes génétiques du récepteur D2 de la dopamine (DRD2) et la tyrosine hydroxylase (TH) sont utiles pour prévoir la réponse à un antidépresseur. Les polymorphismes génétiques sont utiles pour le diagnostic ou pour l'établissement d'un traitement de la dépression au moyen d'un agent antidépresseur, plus particulièrement, un inhibiteur spécifique du recaptage de la sérotonine (SSRI).
PCT/JP2006/318386 2005-09-15 2006-09-15 Polymorphisme genetique utilise pour prevoir une reponse a un agent antidepresseur WO2007032482A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9260757B2 (en) * 2009-10-15 2016-02-16 Life Technologies Corporation Human single nucleotide polymorphisms

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